METHOD FOR QUANTITATIVE PROFILING OF STEROIDS IN HUMAN SALIVA Russian patent published in 2025 - IPC G01N33/74 G01N30/72 G01N33/487 G01N1/40 

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to clinical and laboratory diagnostics, and can be used for quantitative determination of steroids in human saliva, including the following stages: 1) preparation of a synthetic matrix imitating the composition of the native biomaterial, but devoid of detectable endogenous steroid hormones: a) initial matrix is poured into polypropylene test tubes with volume of 50 ml; b) crushed activated carbon is added to the test tubes at rate of 350 mg of coal/15 ml of saliva; c) steroid hormones are absorbed on the surface of activated carbon by mixing the mixture on an orbital shaker for 12 hours at 200 rpm; d) centrifuging tubes for 15 minutes at 13,000 rpm; e) supernatant is transferred into polypropylene test tubes with volume of 50 ml; f) in order to prevent the growth of microorganisms, a preservative – sodium azide is added to the purified matrix at rate of 0.1 g of the preservative/100 ml of the liquid; g) the purified matrix is checked for absence of detected compounds; 2) preparation of solutions of calibrators: Cal1, Cal2, Cal3, Cal4, Cal5, Cal6 and quality control: QCL, QCH and plotting a calibration curve using a synthetic matrix, for which 10 glass test tubes are prepared and marked as follows – BL, Cal1, Cal2, Cal3, Cal4, Cal5, Cal6, QCL, QCH and sample number, in the BL test tube, 500 mcl of the synthetic matrix is added, in Cal1-6, QCL and QCH – 450 mcl of the synthetic matrix and 50 mcl of the solution of the corresponding calibrator or quality control sample, and in a test tube with a sample number – 500 mcl of a human saliva sample; 3) preparation of saliva samples: a) adding 10 mcl of a mixture of internal standards containing methyltestosterone – 250 ng/ml, testosterone-d3 – 250 ng/ml and cortisone-d8 – 250 ng/ml; b) adding 0.5 ml of deionised water and 1.8 ml of ethyl acetate; c) mixing the mixture on a vibration shaker for 10 minutes at speed of 1,000 rpm; d) centrifuging mixture for 5 minutes at a centrifuge rotor speed of 2,500 rpm; e) 1 ml of the organic layer is transferred into 96-well microplate with a well volume of 2 ml; f) evaporating the organic extract to dryness in a stream of nitrogen; g) 150 mcl of a mixture of methanol : water 50:50 vol.% is added to each well of the microplate; h) shaking the contents of wells for 5 minutes at 2,000 rpm, increasing the efficiency of dissolving the dry residue; i) centrifuging mixture for 10 minutes at a centrifuge rotor speed of 2,000 rpm; j) the upper layer of the mixture is transferred into 96-well microplate with volume of 1.1 ml and covered with a silicone mat to ensure the integrity of the sample, as well as its correct supply and dispensing with the appropriate liquid chromatograph module; 4) chromatographic separation of the determined compounds in accordance with tables 1, 2 of the description, followed by mass-spectrometric detection in accordance with table 3 of the description and quantitative determination of steroids in human saliva.

EFFECT: method provides the possibility of increasing the accuracy and rate of quantitative determination of steroids in human saliva due to quantitative determination of steroid hormones in human saliva by high-performance liquid chromatography with mass spectrometric detection in the mode of recording selective transitions with preliminary liquid-liquid extraction with ethyl acetate and using a synthetic matrix.

1 cl, 2 dwg, 5 tbl